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Effects of hexavalent chromium on the adenylate pool of hamster fibroblasts
Insights
Hexavalent chromium (Cr(VI)) disrupts cellular energy by depleting adenosine triphosphate (ATP) levels in hamster fibroblasts. This occurs through inhibiting ATP synthesis and altering nucleotide pools, impacting cell function.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Hexavalent chromium (Cr(VI)) is a known environmental toxicant.
- Understanding its cellular mechanisms, particularly energy metabolism disruption, is crucial.
Purpose of the Study:
- To investigate the mechanisms by which Cr(VI) reduces intracellular adenosine triphosphate (ATP) levels in hamster fibroblasts (BHK 21 line).
- To analyze the effects of Cr(VI) on the adenylate pool composition and ATP biosynthesis.
Main Methods:
- Cells were prelabelled with [3H]adenosine to examine pool component leakage.
- Adenylate pool composition was analyzed using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).
- Endogenous nucleotide concentrations and NAD content were determined by HPLC.
Main Results:
- Cr(VI) treatment led to a significant decrease in ATP, with a concurrent increase in ADP and AMP.
- Radioactive labeling showed altered distribution within the adenylate pool, accumulating in ADP and AMP fractions.
- HPLC analysis revealed reduced NAD content and parallel changes in the guanylate pool.
- Energy charge calculations indicated specific inhibition of ADP phosphorylation to ATP.
- Increased hypoxanthine and inosine in the medium suggested interference with early adenylate synthesis steps.
Conclusions:
- Cr(VI) significantly disrupts cellular energy homeostasis by inhibiting ATP synthesis and altering nucleotide pools in hamster fibroblasts.
- The study identified specific points of interference in ATP biosynthesis and highlighted broader impacts on cellular nucleotide metabolism.
Abstract:
The mechanisms through which Cr(VI) reduces the intracellular levels of ATP in hamster fibroblasts (BHK 21 line) were investigated. Leakage of pool components during treatment of cells prelabelled with [3H]adenosine was examined. Adenylate pool composition was analyzed by thin-layer chromatography (TLC) in cells incubated with [3H]adenosine immediately after treatment with Cr(VI), and the endogenous concentrations of different nucleotides were determined by high-performance liquid chromatography (HPLC). Interference of Cr(VI) with different steps of ATP biosynthesis could be assessed. Treated cells showed increased loss of radioactive hypoxanthine and inosine in the treatment medium, but no nucleotides were detected outside the cells. A marked unbalance of the endogenous adenylate pool was produced by chromium, consisting in a strong decrease of ATP, accompanied by a very pronounced increase of ADP and AMP. Treatment with Cr(VI) caused an altered distribution of label among the different adenylate pool components, especially an accumulation of radioactivity in the ADP and AMP fractions. Also other effects on the soluble pool were detected by HPLC analysis, namely the reduction of NAD content and variations in the guanylate pool which closely paralleled the ones observed in the adenylate pool. Energy charge values, calculates on the basis of HPLC and TLC data, allowed to detect a specific inhibition of ADP phosphorylation to ATP. An interference with the early steps of adenylate synthesis could be inferred from the enhanced amounts of hypoxanthine and inosine in the treated cultures.